This application relates to the field of identification of compounds active on RNA and of target sites on the RNA.
The wealth of information gained from the study of the human genome has allowed identification of many genes whose inappropriate expression results in disease. To exploit this information, several technologies have been developed to specifically target gene expression. Modulating the expression of such genes allows treatment of the disease. In particular, the strategy of modulating gene expression by targeting RNA has spawned several new classes of rationally designed therapeutics (e.g., ribozymes, DNAzymes, and antisense oligonucleotides). However, to date, these technologies have had very limited clinical success. One of the reasons for the limited success is due to the difficult pharmacological barriers these macromolecules must overcome.
The pharmacological treatment of disease is one of the signal advances in medicine in the twentieth century. Modern methods of small molecule development have also proven highly useful in allied fields such as veterinary medicine and pest eradication in agriculture. An ever increasing number of diseases can be ameliorated by appropriate drug therapy, reflecting increasing knowledge of disease pathophysiology and improved methods for conducting clinical research. As the sequence of the human (and other) genomes is solved and the molecular understanding of disease advances it will increasingly be possible to select optimal targets for therapeutic attack. Thus while many current drug treatments act by poorly understood mechanisms, and/or provide only symptomatic relief, increasingly it should be possible to reverse disease pathophysiology and provide more specific and potent therapeutic relief. To achieve this goal, however, will require improved methods of drug development. In particular, the development of drug targeting strategies that enable highly specific targeting of selected genes or gene products will be of great use.
The information provided and references cited herein are provided solely to assist the understanding of the reader, and is not admitted to be prior art to the present invention.
The present invention provides certain advantageous methods for developing biologically active molecules. The invention includes a new method for targeting RNA with small molecules, where the small molecule makes contact with both a cellular protein and a cellular RNA. The effect of the tripartite complex is to increase the affinity of the complex beyond what could be achieved alone with the small molecule and the RNA. This addresses what is believed to be the major limitation of current attempts to target RNA with small molecules, the lack of specific high affinity binding of small molecules to RNAs, which are much more flexible than proteins. This is a generic method for inhibiting any cellular RNA, as described in the disclosure, including, but not limited to allele specific inhibition or inhibition based on targeting to identified regions of RNA secondary structure where that secondary structure differs between allelic forms.
The invention also provides a method for identifying RNA targets for allele specific therapy. These methods have application in at least two major areas: (1) autosomal dominant diseases, or diseases of excess gene dosage, where elimination of one mRNA (but not both) would have a therapeutic effect and (2) cancer and other diseases with LOH where allele specific therapy will provide a differential effect on disease tissue compared to normal tissue. Such use of allele specific therapy has been described in Housman, U.S. Pat. No. 5,702,890, issued Dec. 30, 1997 and Housman et al., U.S. patent application No. 09/045,053, filed Mar. 19, 1998 which are hereby incorporated by reference in their entireties including drawings. Targeting of different alleles can, for example, involve the use of protein-small molelcule complexes as described below, but is not limited to such complexes. For example, different alleles can be targeted with small molecules or with oligonucleotide inhibitors.
The present invention provides a unique strategy for targeting cellular RNAs. This new method overcomes the limitations of current approaches with respect to affinity and specificity. The invention entails formation of a trimolecular complex involving the target RNA, a small molecule, and a cellular protein. The recruitment of a specific cellular protein to the RNA:small molecule complex has the advantage of substantially increasing the area of binding due to contacts between the protein and the RNA, and thereby increase the affinity of the complex beyond what could be achieved alone with the small molecule and the RNA by cooperative binding generally involving electrostatic, hydrogen bonding, and hydrophobic interactions. These favorable binding interactions will increase the affinity of the complex beyond what could be achieved with a small molecule and RNA.
The present invention also provides a new class of RNA therapeutic targets that will provide the basis for inhibition, e.g., allele specific inhibition, by small molecules, and methods for identifying such targets in candidate RNAs. The basis of the invention is the novel observation that single nucleotide polymorphisms in mRNAs are associated with altered secondary structures. These structural differences, not previously known to be associated with normal allelic variation, provide scope for allele specific small molecule inhibition of RNA. Also described are methods for producing and identifying small molecule inhibitors of RNAs.
Thus, in a first aspect the invention provides a method for identifying a potential small molecule inhibitor, where the inhibition involves the induction of formation of a complex of a cellular RNA, a small molecule inhibitor, and a cellular protein. The potential small molecule inhibitor includes a first and a second moiety joined by a linker. The method involves identifying a first moiety which binds to at least a portion of a selected cellular protein; and also identifying a second moiety which binds to at least a fragment of a cellular RNA. Such joint binding indicates that the small molecule is a said potential small molecule inhibitor. The inhibitory ability of the small molecule can be tested by standard methods, e.g., testing the level of protein synthesis from a target mRNA. Clearly, the identifications of the moieties can be performed in either order. The method can also involve selection of a linker or provision of a standard linker, e.g., based on prior screening. In certain cases the linker need not be separate from the first or second moiety, but may be a part of one or both moieties.
In this context, the term xe2x80x9cpotential small molecule inhibitorxe2x80x9d refers to a small molecule which binds to both a selected protein and a target RNA but for which the ability to inhibit a biological activity (e.g., reduce the rate or amount of protein translation from an mRNA) of the RNA has not yet been tested. Following confirmation of such inhibitory characteristic, the small molecule can be referred to as a xe2x80x9csmall molecule inhibitorxe2x80x9d or xe2x80x9cinhibitorxe2x80x9d.
The term xe2x80x9csmall moleculexe2x80x9d refers to a compound which has a molecular mass equal to or less than 5000 Daltons (5 kD), preferably less than 3 kD, still more preferably less than 2 kD, and most preferably less than 1 kD. In some cases it is highly preferred that a small molecule have a molecular mass equal to or less than 700 Da.
The term xe2x80x9cinhibitorxe2x80x9d refers to a compound that reduces to a statistically significant extent at least one activity of a reference compound. In the context of this invention, an inhibitor generally reduces an activity of an RNA, most commonly, but not limited to, reducing or eliminating the rate and/or extent of translation of an mRNA, reducing or eliminating processing of a pre-mRNA to produce a mature RNA; reducing or eliminating transport of an mRNA from nucleus to cytoplasm, or reducing or eliminating a regulatory function of an RNA molecule. Preferably the reduction in activity is sufficient to detect based on gross observation of cellular properties, e.g., growth rate, survival, morphology, and is preferably sufficient and of a nature to produce a therapeutic response in a patient in whom reduction in the expression of the target gene would be beneficial.
In the context of this invention, the phrase xe2x80x9cinduction of formation of a complexxe2x80x9d refers to a property of a small molecule which results in a structure which includes multiple molecules, especially a protein-small molecule-RNA complex, where the components other than the small molecule would not associate with each other or not associate with each other in the same physical relationship to any appreciable extent in the absence of the small molecule or other component which induces formation of the complex. In some cases other molecules may be present in the complex.
In the context of this invention, the term xe2x80x9ctrimolecular complexxe2x80x9d refers to an association or complex which includes a selected protein, a small molecule, and a target RNA. Other components may also be present, e.g., additional proteins or polypeptides, small ions, or water. Preferably, however, the selected protein and the target RNA are the only macromolecules present in the complex. Preferably the stably associated complex consists essentially of or consists of the specified types of molecules.
In preferred embodiments, the identifying of the first moiety involves identifying a known small molecule ligand of the cellular protein. By utilizing a known small molecule ligand (including analogs which retain binding to the protein), it is not necessary to search for a binding moiety, but only to confirm that binding occurs to the selected protein when the ligand is attached to the linker and the second moiety.
In preferred embodiments, the method involves providing a small molecule library containing compounds comprising or consisting essentially of the first moiety and the linker attached to variable second moieties, and screening the library to identify one or more molecules such that the second moiety binds to the target RNA. Preferably the identifying a second moiety involves providing a small molecule library in which compounds have a first moiety which binds to said selected cellular protein, a linker, and a variable second moiety; and screening compounds of the library to identify a compound which binds to the target RNA or fragment thereof. The fragment includes sufficient sequence to retain the characteristics utilized for binding of such molecules to the intact RNA, e.g., local secondary structure.
In reference to selected proteins and target RNA for binding of a first moiety and a second moiety respectively, the terms xe2x80x9cfragmentxe2x80x9d and xe2x80x9cportionxe2x80x9d refer to a part of the sequence of the intact protein or RNA which retains properties of the molecule important for selecting binding. Preferably the fragment or portion retains the local secondary structure as in the intact molecule. For proteins, a portion is preferably at least 10 amino acid residues in length, more preferably at least 20, and still more preferably at least 40 amino acid residues. RNA fragments are preferably at least 20 nucleotides in length, more preferably at least 50 nucleotides in length, and still more preferably at least 100 nucleotides in length.
Reference to xe2x80x9cvariablexe2x80x9d moieties or linkers in a plurality of compounds or molecules or in a library of such compounds means that different molecules in the group have different moieties or linkers as specified.
Conversely, the term xe2x80x9cfixedxe2x80x9d in connection with a moiety or linker in a plurality of compounds (including in a library, e.g., a combinatorial library) means that compounds in the plurality have the same specified moiety or linker.
In preferred embodiments the method involves identifying the first moiety by providing a small molecule library of compounds which have a variable first moiety, a linker, and a fixed second moiety, and screening compounds of the library to identify a compound which binds to the selected cellular protein or portion thereof.
In cases where a known ligand for a selected protein is used, preferably the protein and its ligand are selected from adenosine triphosphate analogs, which could sequester one or more cellular ATPases; flavin adenine dinucleotide or FAD analogs; nicotinamide or nicotinamide analogs; folates or folate analogs, which could sequester proteins such as dihydrofolate reductase, thymidylate synthase or other abundant proteins which require folate as a cofactor; cyclosporin, FK506, rapamycin, and coumermycin or their synthetic analogs, which bind to FK506 Binding Protein (FKBP).
In preferred embodiments, the process of identifying the first or second moieties or the linker involves two or more iterations of at least one of the identification steps. This can be performed to improve the characteristics of the small molecule, e.g., the binding affinity for either or both of the selected protein or target RNA or to improve overall complex stability.
In preferred embodiments, the protein constituent of the trimolecular complex is a cellular protein that is present in the cellular compartment(s) in which the target RNA is present.
Preferably the protein constituent of the trimolecular complex is a protein with known RNA binding activity. Such selection can be advantageous as it is already known that the protein has properties which allow stable interaction with RNA, an interaction which can be utilized in the formation of the desired complex.
Preferably the protein constituent of the trimolecular complex is an RNase., for example an RNase from one of the classes RNaseH type 1, RNaseH type 2, and RNaseL; or an RNA helicase.
In preferred embodiments the protein constituent of the trimolecular complex is an DNA binding protein. Such molecules also provide advantageous nucleic acid binding properties.
In preferred embodiments, the protein constituent of the trimolecular complex has a two or more basic residues within 10 Angstroms of the small molecule binding site thereby providing favorable charge interactions with the target RNA.
As indicated, the molecules are small molecules, which preferably do not exceed 4000 Daltons in molecular mass, more preferably do not exceed 3000 Daltons, still more preferably do not exceed 2000 Daltons, and still more preferably do not exceed 1000 Daltons, or even 700 Daltons.
In preferred embodiments, the targeted cellular RNA is a pre-mRNA or an mRNA. The term xe2x80x9cmRNAxe2x80x9d is used in its usual sense as understood by those skilled in the art. The term xe2x80x9cpre-mRNAxe2x80x9d refers to an RNA transcript which will undergo processing to become an mRNA. Commonly the pre-mRNA contains additional RNA sequence which will be removed to form the mature mRNA.
In preferred embodiments the targeted cellular RNA has at least one polymorphism that results in an altered secondary structure and the altered secondary structure is the target of the RNA binding moiety of said small molecule.
In connection with an RNA, a xe2x80x9csequence polymorphismxe2x80x9d or xe2x80x9cpolymorphismxe2x80x9d is a difference in nucleotide sequence between RNAs transcribed from different allelic forms of a particular gene. Most often, a polymorphism is a single nucleotide substitution. A polymorphism, either alone or in conjunction with one or more other polymorphisms can result in a change in RNA secondary structure, e.g., as shown by changes in digestion with various RNases.
Preferably the targeted cellular RNA is an RNA bearing a mutation that alters secondary structure of the RNA, or that exists on the same allele as another site of variation that alters the secondary structure of the RNA.
The term xe2x80x9callelexe2x80x9d refers to one specific form of a gene within a cell or within a population, the specific form differing from other forms of the same gene in the sequence of at least one, and frequently more than one, variant sites within the sequence of the gene. The sequences at these variant sites that differ between different alleles are termed xe2x80x9cvariancesxe2x80x9d, xe2x80x9cpolymorphismsxe2x80x9d, or xe2x80x9cmutationsxe2x80x9d. The term xe2x80x9calternative allelexe2x80x9d, xe2x80x9calternative formxe2x80x9d, or xe2x80x9callelic formxe2x80x9d refers to an allele that can be distinguished from other alleles by having distinct variances at at least one, and frequently more than one, variant site within the gene sequence.
In preferred embodiments, the method further involves providing a plurality of small molecules which contain a plurality of different linkers and a first moiety identified as described above, or a second moiety identified as described above or both; and screening the plurality of small molecules to identify a potential small molecule inhibitor with advantageous binding or pharmacologic characteristics. The further step of varying the linker can allow selection of a linker which allows more beneficial presentation or association of the protein and the RNA.
In preferred embodiments, the identification of the second moiety involves providing a small molecule library containing compounds containing fixed first moiety and linker attached to variable second moieties; and screening the library to identify one or more molecules in which the second moiety binds to the target RNA. Preferably the identification of the first moiety involves providing a small molecule library of compounds which contain a variable first moiety, a linker, and a fixed second moiety identified as just described; and screening compounds of the library to identify a compound which binds to the selected cellular protein or portion thereof.
In a second aspect, the invention provides a method for identifying an RNA target for an allele specific therapeutic small molecule, e.g., an inhibitor, by identifying at least one sequence variance in the RNA target in a population of interest, determining whether the RNA secondary structure of any sequence variants identified differs between variant alleles. A difference in the RNA secondary structure is indicative that the sequence variance is a potential target for an allele specific therapeutic small molecule.
An xe2x80x9callele specific inhibitorxe2x80x9d or xe2x80x9cvariance specific inhibitorxe2x80x9d is a drug or inhibitor that inhibits the activity of one alternative allele of a gene to a greater degree than at least one other alternative allele. The difference in activity is commonly determined by the dose or level of a drug required to achieve a quantitative degree of inhibition. A commonly used measure of activity is the IC50 or concentration of the drug required to achieve a 50% reduction in the measured activity of the target gene.
Preferably an allele specific inhibitor will have at least twice the activity on the target allelic form than on a non-target allelic form, more preferably at least 5 times, still more preferably at least 10 times, and still more preferably at least 50 times, and most preferably at least 100 times. This can also be expressed as the sensitivities of the different allelic forms to the inhibitor. Thus, for example, it is equivalent to state that the target allelic form is most preferably at least 100 times as sensitive to the inhibitor as a non-target allelic form. The activity of an inhibitor can be measured either in vitro or in vivo, in assay systems that reconstitute the in vivo system, or in systems incorporating selected elements of the complete biological system. For use in inhibiting cells containing only the target allelic form rather than cells containing at least one non-targeted allelic form, the difference in activity is preferably sufficient to reduce the proliferation rate or survival rate of the cells having only the targeted allelic form to no more than one half of the proliferation rate or survival rate of cells having at least one non-targeted allelic form. More preferably, the fraction is no more than ⅕ or {fraction (1/10)}, and still more preferably no more than {fraction (1/20)}, {fraction (1/50)}, {fraction (1/100)}, or even lower.
In this context, the term xe2x80x9cpopulationxe2x80x9d refers to a group of individuals (generally a group of humans) distinguishable from other groups. Bases for distinguishing can, for example, include geography, familial relationship, racial background, age, gender, and combinations of these factors. A xe2x80x9cpopulation of interestxe2x80x9d is thus a group of individuals which are relevant to identification of a target, a therapeutic or complex-forming agent, or a method of using or making such agents or other aspect of this invention, e.g., a group to which a patient belongs in most cases a population will preferably encompass at least ten thousand, one hundred thousand, one million, ten million, or more individuals, with the larger numbers being more preferable. In special circumstances, diseases will occur with high frequency in specific geographical regions or within specific familial, racial, or cultural groups, and a relevant population may usefully be considered to be a smaller group.
Preferably, the sequence variance to be targeted occurs in an allele frequency range between 0.1:0.9 and 0.5:0.5 in a population of interest.
The term xe2x80x9callele frequencyxe2x80x9d refers to the fraction (or frequency of occurrence) of a specific allele as compared to all alleles in a population. It is recognized in the art that the heterozygote frequency and allele frequency are related and, for certain alleles, can be described by Hardy Weinberg equilibrium calculations. It will also be recognized that sequence variances that occur at high frequency in the population are commonly not deleterious to the health of the individuals who carry these genes and are commonly not disease genes or mutations that are associated with disease.
In preferred embodiments, the determination of the RNA secondary structure is performed using one or more structure-specific enzymes, e.g., a nuclease, preferably selected from T1, T2, S1, U2, CL3, V1, A, PhyM, N.c. nuclease or RNase; or by chemical probing with a chemical selected from the group consisting of dimethyl sulfate, diethylpyrocarbonate, CMCT, kethoxal, bisulfite, ethylnitrosourea, MPE-Fe(II), and Fe(II)-EDTA.
Allele specific targeting can advantageously be applied in a case where the target RNA is an allele that, if reduced in abundance, would at least ameliorate a disease state. The amelioration can, for example, be symptomatic relief, reduction or elimination of one or more deleterious effects of the disease, or even cure of the disease.
Likewise, the target RNA can be an allele associated with an autosomal dominant disease, such as Huntington""s disease, or other diseases shown in Table 1.
The target RNA can also be an allele of an essential gene which undergoes loss of heterozyosity in a proliferative disorder, e.g., a cancer. For example, the targets identified by the methods herein can advantageously be utilized in the therapeutic approach described in Housman, supra.
The term xe2x80x9cproliferative disorderxe2x80x9d refers to various cancers and disorders characterized by abnormal growth of somatic cells leading to an abnormal mass of tissue which exhibits abnormal proliferation, and consequently, the growth of which exceeds and is uncoordinated with that of the normal tissues. The abnormal mass of cells is referred to as a xe2x80x9ctumorxe2x80x9d, where the term tumor can include both localized cell masses and dispersed cells, The term xe2x80x9ccancerxe2x80x9d refers to a neoplastic growth and is synonymous with the terms xe2x80x9cmalignancyxe2x80x9d, or xe2x80x9cmalignant tumorxe2x80x9d. The treatment of cancers and the identification of anticancer agents is the concern of certain preferred embodiments of the aspects of the present invention. Other abnormal proliferative diseases include xe2x80x9cnonmalignant tumorsxe2x80x9d, and xe2x80x9cdysplasticxe2x80x9d conditions including, but not limited to, leiomyomas, endometriosis, benign prostate hypertrophy, atherosclerotic plagues, and dysplastic epithelium of lung, breast, cervix, or other tissues. Drugs used in treating cancer and other non-cancer proliferative disorders commonly aim to inhibit the proliferation of cells and are commonly referred to as antiproliferative agents.
In preferred embodiments, the allele specific small molecule is a small molecule inhibitor containing a first moiety which binds to a selected cellular protein or a portion thereof, a second moiety which binds to a target cellular RNA or a fragment thereof, and a linker connecting the first moiety and the second moiety, where the small molecule inhibitor can bind to the selected cellular protein and to the target cellular RNA to form a complex. The molecule can be as further described for those identified in the first aspect above and for corresponding pharmaceutical compositions and complex-forming molecules.
In a third aspect, the invention provides a method for identifying an allele specific RNA binding small molecule for a target RNA, involving the steps of identifying at least one sequence variance in the RNA target in a population of interest having secondary structure differences between variant alleles, screening the two RNA structures for a said sequence variance or variances for binding to or inhibition by a plurality of small molecules; and identifying a compound that preferentially binds to or inhibits one of said RNA structures, where the binding or inhibition is indicative that the compound is an allele specific RNA binding small molecule. Preferably the plurality of small molecules are elements of a small molecule combinatorial library.
Preferably the method includes determining whether the allele specific RNA binding small molecule provides a cellular activity, by contacting cells producing the target RNA with the allele specific RNA binding small molecule; and determining whether the small molecule produces a change in cellular activity. Thus, the method uses a cellular assay to identify cellular effects. Preferably the cellular effect or activity is a decrease in expression of the target RNA or the translation product.
As in preceding aspects, the target RNA is preferably a pre-mRNA or an mRNA.
In preferred embodiments, the plurality of small molecules is a plurality of potential small molecule inhibitors each comprising a first moiety which may be the same or different and a second moiety which differs between the plurality of small molecules and a linker joining the first moiety and the second moiety. The first moiety is selected to bind to a selected-cellular protein or a portion thereof; and the second moiety is selected to bind to a target cellular RNA or a fragment thereof, thereby forming a trimolecular complex.
Additional preferred embodiments are as described for the first aspect above.
In a fourth aspect, the invention provides a pharmaceutical composition which includes a small molecule inhibitor where the inhibitor includes a first moiety which binds to a selected cellular protein or a portion thereof; a second moiety which binds to a target cellular RNA or a fragment thereof, and a linker connecting said the moiety and the second moiety, where the small molecule inhibitor can bind to the selected cellular protein and to the target cellular RNA to form a complex. The composition also includes a pharmaceutically acceptable carrier, excipient, or diluent. Generally the inhibitor will be a synthetic molecule.
In the context of the inhibitor or binding compounds of this invention, the term xe2x80x9csyntheticxe2x80x9d indicates that the compound has been prepared in whole or in part by the intervention of humans, and is not a compound naturally produced by a naturally occurring organism. A portion or even most of a synthetic molecule can be produced by an organism, and then modified by synthetic methods. Alternatively, most or all of a molecule can be prepared by synthetic methods, and the resulting molecule is then referred to as xe2x80x9cfully syntheticxe2x80x9d.
In certain cases, a naturally occurring molecule may be found which has the complex-forming properties described. Such a molecule could be used in the methods described herein for using compounds which inhibit or bind RNA, or could be modified to alter its binding and/or activity characteristics.
Preferably the selected protein is a human protein; preferably the target RNA is a human RNA.
Preferably the small molecule inhibitor inhibits expression of said target RNA.
In preferred embodiments the small molecule inhibitor has differential activity on variant forms of the RNA differing in secondary structure, where the difference in secondary structure is related to at least one sequence variance. Preferably the targeted cellular RNA has a polymorphism that results in an altered secondary structure and the altered secondary structure is the target of the RNA binding moiety of said small molecule.
Also in preferred embodiments, the inhibitor is a compound as identified in the first aspect above and/or the target is as identified in the second aspect.
In a fifth aspect, the invention provides a method for inhibiting expression of an mRNA. The method involves contacting cells normally expressing the mRNA with a small molecule inhibitor. The inhibitor has a first moiety which binds to a selected cellular protein or a portion thereof, a second moiety which binds to a target cellular mRNA, and a linker connecting the first moiety and the second moiety. The small molecule inhibitor can bind to the selected cellular protein and to the target cellular mRNA to form a complex.
In preferred embodiments, the molecule and/or target is as described for embodiments of aspects above.
In a sixth aspect, the invention provides a method for treating an autosomal dominant or gene dosage disease, by administering to a patient suffering from such a disease a therapeutically effective amount of an allele specific RNA inhibitor preferentially active on an allelic form of a target RNA in which at least one sequence variance provides a difference in secondary structure and wherein the allele specificity involves that difference in secondary structure.
In preferred embodiments, the disease is an autosomal dominant disease, and the patient is heterozygous for the gene corresponding to the target RNA.
Also in preferred embodiments, the disease is a gene dosage disease, and said administering is adjusted to reduce but not eliminate expression of the gene. Preferably the patient is heterozygous for the gene and the inhibitor preferentially reduces or eliminates expression of only one allele.
The term xe2x80x9cgene dosage diseasexe2x80x9d refers to a disease which is at least partially has a cause based on overactiyity of a product of a gene, e.g., overexpression of the gene.
A xe2x80x9ctherapeutic effectxe2x80x9d results, to some extent, in a measurable response in the treated disease or condition. Thus, a therapeutic effect can, for example, include a cure, or a lessening of the growth rate or size of a lesion such as a tumor, or an increase in the survival time of treated patients compared to controls, a relief of a deleterious symptom, or a slowing of the progression of a disease, among other possible effects.
The term xe2x80x9ctherapeutic amountxe2x80x9d means an amount which, when administered appropriately to a patient e.g., a mammal, e.g., a human, suffering from a disease or condition, produces a therapeutic effect.
In preferred embodiments, the molecule and/or target is as described for aspects and embodiments above.
In a seventh aspect, the invention provides a method of treating a patient suffering from a disease or condition, by administering to the patient an allele specific inhibitor of a target RNA where the allele specificity involves a difference in secondary structure related to at least one sequence variance.
In preferred embodiments, the method involves determining the presence of the at least one sequence variance in the patient.
In preferred embodiments, the inhibitor and/or the target is as described for aspects or embodiments above.
In an eighth aspect, the invention provides a method for producing a pharmaceutical agent, by identifying a small molecule inhibitor of a cellular RNA. The inhibitor induces formation of a complex with a cellular protein and the inhibitor includes a first moiety and a second moiety and a linker joining the first moiety and the second moiety. The method involves identifying a first moiety which binds to a selected cellular protein or a portion thereof; and identifying a second moiety which binds to a target cellular RNA or a fragment thereof, and synthesizing the small molecule inhibitor in an amount sufficient to provide a therapeutic response when administered to a patient. The identification can also involve selecting a linker which allows advantageous complex formation.
In preferred embodiments, the inhibitor is preferentially active on an allelic form of an mRNA in which at least one sequence variance provides a difference in RNA secondary structure.
Further preferred embodiments include embodiments as specified in the first and/or second aspects.
In the methods of inhibiting an RNA and/or methods of treating a patient, a compound may be used in various ways, for example, the compound may be used alone, or may be used as part of a pharmaceutical composition.
In a ninth aspect, the invention provides a complex-forming molecule which includes a first moiety which binds to a selected cellular protein or a portion thereof; a second moiety which binds to a target cellular RNA or a fragment thereof, and a linker connecting said first moiety and said second moiety. The small molecule inhibitor can bind to the selected cellular protein and to the target cellular RNA to form a complex.
In preferred embodiments the molecule will inhibit expression of a target mRNA under translation conditions.
xe2x80x9cTranslation conditionsxe2x80x9d refers to conditions such that translation would occur from a particular selected mRNA in the absence of an inhibitor of translation. Thus, such conditions can include where an in vitro translation system is present as well as in vivo conditions.
In preferred embodiments, the selected cellular protein is an RNA binding protein or a derivative thereof.
Further preferred embodiments are as described for the inhibitors in pharmaceutical compositions herein and/or has components, properties, targets or binding pairs as otherwise described herein.
By xe2x80x9ccomprisingxe2x80x9d is meant including, but not limited to, whatever follows the word xe2x80x9ccomprisingxe2x80x9d. Thus, use of the term xe2x80x9ccomprisingxe2x80x9d indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By xe2x80x9cconsisting ofxe2x80x9d is meant including, and limited to, whatever follows the phrase xe2x80x9cconsisting ofxe2x80x9d. Thus, the phrase xe2x80x9cconsisting ofxe2x80x9d indicates that the listed elements are required or mandatory, and that no other elements may be present. By xe2x80x9cconsisting essentially ofxe2x80x9d is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase xe2x80x9cconsisting essentially ofxe2x80x9d indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.
Other features, advantages and embodiments will be apparent from the following Detailed Description of the Preferred Embodiments and from the Claims.